(1) Mechanisms of virulence acquisition in Ngari virus (NRIV): In 1997-98 a large outbreak of Rift Valley fever (RVF) occurred in Kenya and Somalia. During this outbreak, NRIV was identified as the causative agent of hemorrhagic fever (HF) in a significant proportion of the cases. NRIV was also recognized as a naturally occurring genetic reassortant between Bunyamwera virus (BUNV; L and S segments) and Batai virus (BATV; M segment), both of which belong to the bunyamwera serogroup in the genus Orthobunyavirus. Interestingly, both parental viruses cause febrile illness, but not severe HF in humans. This is a perfect example of the important role that genetic reassortment plays in the evolution of viruses and of the changes in virulence that can result. Therefore, we are using NRIV as a model to understand the molecular mechanisms underlying the emergence of novel pathogenic bunyaviruses in nature. So far, we have determined the full genome sequences of 5 NRIV isolates (genotype: L-BUNV/M-BATV/S-BUNV), including 2 strains from the Kenya/Somalia HF outbreak, as well as 2 BUNV isolates and 5 BATV isolates. Based on analysis of these data we have made several interesting observation related to the emergence of NRIV: 1) that genetic variation among NRIV strains is low, suggesting that a recent single reassortment event may have given rise to all the identified NRIV isolates and 2) that while overall genetic diversity among NRIV strains is low, a significant amount of this total variation is present in strains from a single outbreak, suggesting that multiple introductions into the human population are responsible for the large outbreaks seen with NRIV. In addition to these direct observations, the availability of these sequences also provides us with the possibility to pursue full-length clone generation for these viruses, work that is currently in progress. In future these systems will allow us to construct various reassortant and chimeric viruses and thereby map the viral determinants associated with NRIV virulence and its ability to cause HF. Further, in order to identify measurable phenotypic characteristics that could be related to the acquisition of virulence by NRIV we have been examining growth in various cell lines, including both human and mosquito cells. Interestingly we have determined that while in a highly permissive cell line (Vero) all BATV strains grow well and with similar kinetics, in C6/36 (mosquito cells) only a few BATV strains, including the UgMP-6830 strain, which is the closest relative of the NRIV M-segment, show efficient growth at early time points after infection. This could have implications for successful virus spread in the mosquito vector and potentially limit which BATV strains can generate NRIV-like viruses during reassortment. In addition, we have preliminary data demonstrated enhanced growth of BUNV and NRIV strains over BATV strains in primary human macrophages. This finding is currently being followed up and will include the analysis of cytokine production in response to infection with these different viruses, since for other hemorrhagic fever viruses aberrant cytokine responses have been shown to be critical in the development of disease. In addition, the differences observed in virus growth among different cell lines promoted consideration that these viruses might be using distinct receptors. To examine this we have generated soluble version of the receptor-binding subunit of BUNV, BATV and NRIV. However, while we can show that these proteins bind to target cells, in fibroblasts they block infection with all three viruses equally well, indicating that in this cell type receptor usage is overlapping. Studies to examine if this differs in putative primary target cells like macrophages are currently on-going. (2) Molecular determinants of host range in Simbu serogroup viruses: The simbu serogroup also belongs to the genus Orthobunyavirus and can be categorized into those viruses that infect i) humans (Oropouche virus), ii) livestock animals (e.g. Akabane virus) or iii) vertebrate hosts (rodents, birds, monkey etc.) other than humans or livestock (e.g. Mermet virus). Among the simbu-serogroup viruses Oropouche virus (OROV) is unique in causing acute febrile illness in humans. Since its discovery, OROV has caused more than 30 outbreaks with at least 500,000 cases identified between 1960 and 2009 in South America. It is currently unknown why OROV causes disease in human but other simbu viruses do not. In order to identify the viral determinants of host range among simbu serogroup viruses, we will compare the genetic and virological characteristics of OROV and other related viruses, especially those that infect non-livestock animals, since these viruses are genetically closer to OROV than those that infect livestock. To accomplish this, we have determined the full genome sequence of over 25 simbu serogroup viruses. Our analyses have revealed that simbu group viruses that infect vertebrate hosts other than humans or livestock can be divided genetically and geographically into two groups, Old World and New World. In addition, we have identified several South American simbu group viruses that are genetically closely related to the human pathogenic OROV. Interestingly, we found that one of these OROV-like virus, Utinga virus, which is not known to cause illness in humans but was isolated from the same animal reservoir as OROV (sloths), does not encode the non-structural protein NSs, which inhibits the host interferon antiviral response. Based on these results, we have started to analyze the molecular and biological properties of New World simbu group viruses and are attempting to develop reverse genetics systems for OROV to study its molecular biology and pathogenesis. (3) Molecular characterization of uncharacterized orthobunyaviruses and taxonomically ungrouped bunyaviruses potentially causing human disease: In order to better understand the relationships between the molecular biological characteristics of uncharacterized viruses and their zoonotic potential, as well as their evolution, we will conduct an extensive genetic analysis and biological characterization of uncharacterized orthobunyaviruses and taxonomically ungrouped bunyaviruses isolated from Africa, Asia, South and North America. So far, we have determined the genome sequences of around 70 bunyaviruses, including genetically uncharacterized orthobunyaviruses and taxonomically ungrouped bunyaviruses. Our extensive genome sequencing attempts have identified Bhanja virus (BHAV), which was isolated from a tick collected in India, as belonging to a novel species in the genus Phlebovirus. Based on our genetic characterization of the BHAV genome we could determine that BHAV has the highest identity to severe fever with thrombocytopenia syndrome virus (SFTSV), a novel phlebovirus first recognized in 2009 as the cause of a tick-borne hemorrhagic fever-like syndrome in humans in China. Serological analyses also indicate that BHAV is serologically related to SFTSV. These results suggest that BHAV will be classified as a novel species in the genus Phlebovirus and that it constitutes the missing link between the newly identified human pathogen SFTSV and other tick-borne phleboviruses. Sporadic human cases of BHAV and genetic similarities of BHAV to SFTSV suggest pathogenic potential of BHAV for humans. Our sequence data and ongoing molecular characterization of BHAV will facilitate epidemiological and epizoological surveillance for BHAV infections as a potentially under-recognized cause of acute febrile illness.